gms | German Medical Science

Objective: Staphylococcus aureus is the most frequent causal agent for bone infection. When S. aureus infection affects a total joint arthroplasty, removal and replacement of joint is mostly required. Moreover, it was recently discovered that S. aureus is able to invade osteoblasts thus escaping the extracellular host antibacterial defense and even antibiotics. This plays a significant role in persistence and recurrence of infection. The aim of this study was to investigate whether (1) S. aureus is able to not only invade but also proliferate within osteoblasts, (2) the mechanism of invasion and (3) to clarify whether rifampicin or gentamicin can inhibit intracellular proliferation and survival of S. aureus.

Method: SAOS-2 osteoblast-like cell line were grown in Minimum Essential Medium and subsequently infected with S. aureus EDCC5055 and S. aureus Rosenbach 1884 (ATCC 12598). In order to test the mode of bacterial internalization, SAOS-2 cells were treated 2h prior to infection with cytochalasin D which is the major actin depolymerization agent disrupting actin microfilaments. Immunofluorescence and transmission electromicroscopic (TEM) imaging were performed to detect potential intracellular and proliferating bacteria. For antibiotic experiments, SAOS-2 osteoblasts infected with S. aureus were treated with 7.5 microgram/ml of rifampicin or 30 microgram/ml, 100 microgram/ml, or 200 microgram/ml of gentamicin for 4h and 24h.

Results and conclusion: Both S. aureus strains were able to efficiently invade and to proliferate within human osteoblasts shown by typical bacterial growth curves. Immunofluorescence microscopy showed intracellular invasion of S. aureus and TEM images could demonstrate bacterial division as well as disruption of lysosomal membranes as a sign of successful intracellular proliferation and survival. Cytochalasin D was able to significantly reduce S. aureus invasion ability suggesting that invasion was enabled by promoting actin rearrangement at the cell surface. 7.5 microgram/ml of rifampicin was able to inhibit bacterial survival in human osteoblasts with almost complete elimination of bacteria after 4 h. Effects of gentamicin were dose-dependent but even high doses with 200 microgram/ml of gentamicin were associated with a statistically significant higher number of survived bacteria compared to rifampicin. In conclusion, S. aureus is not only able to invade but also to proliferate in osteoblasts. Invasion seems to be associated with actin rearrangement at the cell surface. Rifampicin is effective in intracellular eradication of S. aureus whereas gentamicin seems to have a much weaker intracellular effect. Based on these data, doses of rifampicin and gentamicin could be optimized for local use to coat end prostheses and be able to reach the intracellular compartments thus killing the probable intracellular persisting S. aureus without causing undesirable systemic side effects.